About 40% of hyperthyroidism patients suffer from Graves' disease (Morbus Basedow), an autoimmune disease in which autoantibodies activate the thyrotropin receptor, mimicking its natural hormone ligand, the thyroid-stimulating hormone (TSH). This pathological activation of TSH-Receptor (TSHR) leads to uncontrolled production of thyroid hormones causing hyperthyroidism. TSH and the TSHR are key proteins in the control of thyroid function. In fact, TSHR is mainly expressed in follicular epithelial cells of the thyroid gland, but also in a variety of additional cell types such as retro-orbital fibroblasts, kidney, adipocytes and bone cells. TSH binds to its receptor and leads to the stimulation of second messenger pathways involving predominantly cAMP. Inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) pathways are also activated at higher TSH concentrations.
The treatment of choice applied in the clinics for decades involves thyrostatic drugs blocking the production of thyroid hormones (TH). This medication plays a role further downstream in the signal cascade of the thyroid upon activation of the TSHR. Since thyroid hormones T3, T4 are secreted in the thyroid gland and these medications induce an inhibition of their synthesis. Thus, the current primary anti-thyroid treatment does not target the causative molecular activation of the TSHR by antibodies and patients are therefore burdened by a rate of at least 5% adverse effects (Sato et al. 2014). This demands frequent controls of the thyroid hormone levels and adjustments of thyrostatics dosage. In contrast to these drugs, which regulate the thyroid hormone level, the most promising target is the TSHR itself. However, small allosteric antagonists acting directly at the TSHR are not available on the market yet.
Therapeutic Gap for Graves' Ophthalmopathy
Moreover, about 25% of Graves' disease patients also develop an orbitopathy referred to as “Graves' Ophthalmopathy” (GO), a related organ-specific autoimmune disease affecting the appearance and functioning of the eyes. Progression to severe forms occurs rarely, no more than 3% to 6% of cases, however available therapies (Eckstein et al. 2012) are largely imperfect and remain still a dilemma (Bartalena 2011). There is considerable evidence that expression of the TSHR in the orbital fibroblasts (OF) and orbital adipocytes behind the eye may contribute to this difficult-to-treat orbitopathy, and thyroid stimulating antibodies titer tend to correlate with severity of GO. Orbital fibroblast have been recognized as primary target cells of autoimmune attack and TSHR acts as a primary autoantigen in GO. The pathological activation of TSHR leads to the production of the extracellular matrix by involvement of hyaluronan acid (HA), fibrosis and swelling of extraocular muscle as well as adipogenesis of orbital fibroblasts (orbital fat expansion) (Sorisky et al. 1996; Feliciello et al. 1993). The increase in tissue volume in the orbit often causes diplopia and compression of the optic nerve and exophthalmos. Human retroocular fibroblasts have been utilized as in vitro models to study GO.
Apart from glucocorticoids and the IGF-1 receptor, which is also considered as antigen in GO, the TSHR is reckoned to be a potential target for pharmacological intervention of GO which is due to the following facts: TSHR expression is increased with adipogenesis, the TSAb (M22) itself enhances adipogenesis, it has been shown that the TSHR is linked with the HA production, since the M22 stimulated HA synthesis can be inhibited by a PI3K inhibitor or by an inhibitor of mTOR, and different TRAb induce divergent signaling pathways downstream of the TSHR such as c-Raf-ERK via Gq as unique cascade, which is not activated by TSH, which primarily activates Gs.
Taken together autoimmune autoantibodies are activating TSHR in the orbita as well, but in contrast to the thyroid different molecular mechanisms are activated. Therefore, the anti-thyroid drugs available on the market blocking the thyroid hormone synthesis in the thyroid are non-effective for treatment of thyroid eye disease. This unveils the therapeutic gap for the treatment of severe GO patients in the clinics. This apparent problem has been repeatedly expressed at the annual international congresses of the European Thyroid Association (Bartalena 2013).
Pharmacological Approaches to Tackle Graves' Ophthalmopathy
A potential approach for the therapy of GO could be the suppression of the pathological activation caused by autoantibodies directly on the TSHR by drug like small molecule ligands (SM). Thus orally active small allosteric antagonists have a strong therapeutic potential underlining their therapeutic importance for TSHR-mediated GO. In contrast to the activating autoantibodies that bind like the TSH to the extracellular region of the TSHR, such synthetic SMs preferentially bind elsewhere, for example allosterically into a binding pocket located within the heptahelical transmembrane domain of the receptor (FIG. 1).
Presently some success has been obtained by the application of antibodies that block TSHR activation, which bind similarly to TSH at the extracellular binding site of the TSHR. Crystal structures of activating (M22) and blocking (K1-70) monoclonal antibodies identified the different interacting residues at the extracellular leucine rich repeat domain (LRRD) for both types. Immunomodulation is also considered a feasible approach to tackle GO. However, for example introducing an antibody for immunomodulation as a potential drug will be difficult to produce in large quantities with uniform quality and it is in magnitudes more cost-intensive than small molecules.
Analysis of Allosteric Transmembrane Binding Pocket at TSHR
Together with the lutropin receptor (LHCGR) and follitropin (FSH) receptor (FSHR), the TSHR (Kleinau & Krause 2009) belongs to the subfamily of glycoprotein-hormone receptors (GPHRs) within the rhodopsin/adrenergic receptor family 1 of the GPCR superfamily. A special feature of GPHRs is the very large extracellular portion, where the hormone binds between a leucine rich repeat domain (LRRD) and a hinge region and thereby the activation is initiated (FIG. 11).
For the TSHR the functional and structural dimensions of its extracellular region were defined and an intramolecular agonistic unit was suggested (Kleinau, Mueller, et al. 2011). The signal is then conveyed through the heptahelical transmembrane domain into the cell via the heterotrimeric G-protein. It has been shown that the developed small agonist compound 2 (FIG. 1) is interacting in the transmembrane binding pocket (Neumann et al. 2009). The allosteric binding pocket was investigated in more detail by modelling driven mutagenesis of about 30 mutations. This led to distinct constitutively activating mutations (CAM) by V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F L645V, Y667A (G. Kleinau et al. 2010) and silencing mutations V4241, L467V, Y582A, Y582F, Y643A, L665V (Haas et al. 2011) indicating not only key amino acids covering the allosteric binding pocket at the TSHR but also positions, where the TSHR conformation can be changed to an active or inactive state respectively.
Allosteric Modulators for the GPHRs, Focusing on the TSHR
One low molecular weight agonist for a GPHR, org41841, was reported as selective for the lutropin receptor LHCGR (van Straten et al. 2002). It was shown that org41841 is also a partial agonist for the TSHR and it binds allosterically into a transmembrane binding pocket (Jäschke et al. 2006). The identification and characterization of an additional binding pocket located in the transmembrane domain of GPHRs led to the development of drug like small molecules targeting this family. An antagonistic compound (NIDDK/CEB-52 (c52)) was developed, albeit with low affinity at TSHR, exhibiting no effects at FSHR but partial agonistic activity at LHCGR (G Kleinau et al. 2008).
A high-throughput screen (HTS) for TSHR agonists (Titus et al. 2008) together with structure-functional analyses led to the TSHR agonists described in WO/2010/047674 and the identification of the first nanomolar TSHR selective agonist named ‘compound 2’, (Neumann et al. 2009). Modifications of compound 2 led to an inverse agonist (S2-7) with micromolar affinity for the TSHR, which inhibits basal signaling at wild type and at four constitutively active mutants of TSHR (Neumann et al. 2010). This antagonistic compound was also tested on orbital fibroblasts involved in pathogenesis of GO (Turcu et al. 2013) and in mice (Neumann et al. 2014). However, one has to note that this compound exhibits a cross-reactivity towards the FSHR.
The tetrahydroquinoline compound Org274179-0, was reported as a nanomolar TSHR antagonist (van Koppen et al. 2012), but exhibits cross-reactivity for the FSHR and LHCGR as well. This fact is not surprising as the tetrahydroquinoline scaffold is a derivative of the strong FSHR antagonist previously developed by the former company Organon (van Straten et al. 2005). For this reason therapeutical application of this substance type is disadvantageous, since the side effects by blocking the FSHR activation, resulting for example in reduced spermatogenesis in men, would be unfavorable. Taken together, according to the best knowledge of the inventors there is no TSHR antagonist described previously in clinical use, and only two other small molecules with antagonistic effects at TSHR (S2-7, Org274179-0) have been published, but both with cross reactivity to FSHR, albeit with different strengths.
Chemical compounds with nuclear hormone modulatory function have been disclosed in the art. WO 2002/067939 discloses fused cyclic succinimide compounds and analogs thereof as modulators of nuclear hormone receptor function, in particular as modulators of the androgen receptor (AR), the estrogen receptor (ER), the progesterone receptor (PR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), the aldosterone receptor (ALDR) and the steroid and xenobiotic receptor (SXR), potentially for use in the treatment of autoimmune thyroiditis. No disclosure is evident that such compounds exhibit a TSHR antagonistic activity suitable in treating hyperthyroidism.
Atamanyuk et al. (Journal of Sulfur Chemistry, 2008, 29:2, p 151-162) disclose an anti-cancer activity of thiopyrano(2,3-d)thiazole-based compounds containing a norbornane moiety. Lesyk et al. (Biopolimery I Kletka, 2011, 27:2, p 107-111) disclose 4-thiazolidinones and related heterocyclic compounds and their anti-cancer activity. No mention is made in either document of a TSHR antagonistic activity of such compounds that would be suitable in treating hyperthyroidism.
In light of the prior art there remains a significant need in the art to provide additional means for the treatment of hyperthyroidism, in particular for providing compounds that act as TSHR antagonists.